It is well known that a nucleic acid such as deoxyribonucleic acid (DNA) is able to serve as its own template during self-replication. It is also well known that a double stranded or duplex nucleic acid can be separated into its component single strands. These properties have been exploited to permit the in vitro amplification and modification of nucleic acid sequences by the polymerase chain reaction (PCR) (also referred to herein as two-primer amplification).
PCR is an in vitro, enzyme-based replication of nucleic acid sequences, using two oligonucleotide primers designed to hybridize to opposite strands and flank the region of interest on the target polynucleotide sequence. During repetitive cycles the nucleic acid is subjected to strand separation, typically by thermal denaturation, the primers are hybridized (by annealing if thermal cycling is used) to the single strand templates, and an enzyme such as DNA polymerase (DNA template to DNA primer extension) or reverse transcriptase (ribonucleic acid or "RNA" template to DNA primer extension or DNA template to DNA primer extension) extends the primers on the templates. Both of the strands (plus and minus), including newly synthesized strands, are made available as templates for the extension of both primers respectively by the strand separation step. The result, with two primers, is an exponential increase (hence the term "chain reaction") in template nucleic acid copy number (both plus and minus strands) with each cycle, because with each cycle both the plus and minus chains are replicated (1, 2). The nucleic acid duplex which results will have termini corresponding to the ends of the specific primers used. It is possible, by means of PCR, to amplify, detect, or otherwise modify a nucleic acid sequence in vitro (1, 2).
The art teaches that if a single unpaired primer is used in place of two (paired) primers, the result is a linear growth in extension product copy number instead of an exponential growth of both strands (3). It is generally believed that the reason for the linear growth in copy number with cycle number using a single unpaired primer is that only the template strand is replicated during each cycle. The primer extension itself is not copied.
The linear growth in copy number with a single primer was confirmed by Kim et al. (3). Kim et al. developed a recombinant fragment assay based on PCR amplification. A pair of primers were prepared which were each complementary to the opposite terminals of the recombinant sequence expected to be formed from two parent chromosomal sequences. Each of the two parent chromosomal sequences was complementary to only one of the primers. As predicted, only the recombinant sequence (having binding sites for both primers) was detected by Kim et al. after 50 cycles. Thus, this assay provides a strong confirmation for the previous observations in the art literature (1,2,3,4) that two primers are required for exponential amplification.
Single primer amplification has been used to perform "cycle sequencing" in a process marketed by Applied Biosystems, Inc. (San Jose, Calif.). Cycle sequencing relies upon a single, dye labeled primer or terminator to achieve linear amplification of the extension products. The primer extension products are then collected and sequenced in order to derive the sequence of the original DNA template. This technique is reported to be particularly advantageous in allowing sequencing of large constructs with minimal sample size (e.g. 42 kb construct using only 1.2 .mu.g of sample) (5). However, the amplification achieved during cycle sequencing is described as different from PCR because "PCR uses two different primers to achieve exponential amplification of the template . . . . Cycle sequencing, on the other hand, uses only one primer to achieve linear amplification of the extension products." (5).
Single-primer linear amplification has also proved useful as a technique for detecting DNA methylation and protein-DNA interactions by providing "selective, linear amplification by thermostable DNA polymerase from Thermus aquaticus" (taq DNA polymerase) (6). Thus, single-primer amplification is used by the art in certain specialized procedures to provide the expected linear amplification.
The preparation of primers for PCR requires that the terminal sequences of the nucleic acid strands (both the plus and minus templates) to be amplified or detected, be known (2). The sequence information may be derived by direct sequencing of the terminals of the nucleic acid of interest, or by sequencing the terminal of a polypeptide and producing a corresponding copy oligonucleotide primer. The optimal primer size is typically about 20-30 bases in length (2), but workable primers may be smaller or larger in particular circumstances. As is well known, as primer size decreases, the likelihood that the primer will hybridize to an unplanned site on the sequence of interest increases. Unplanned hybridizations can lead to an interruption of amplification of the desired product and production of products having either a smaller size or an undesired primer insert. Thus, the selection of two optimal primers for PCR requires the avoidance of unplanned hybridization with the sequence of interest whenever practicable.
The rational selection of primer sequence to avoid unplanned hybridizations is well known. Algorithms are known by which the artisan may compare proposed primer sequences to the entire template sequence (where known) and to any other sequences which are known to be present in an assay mixture. Such algorithms are typically implemented by means of a programmable digital computer able to store sequences for comparison, execute a programmed comparison of all sequences, and thereby estimate the likelihood of a desired or an unplanned hybridization occurring based upon a determination of relative percent complementarities and other factors known to affect the likelihood of hybridization (e.g. stringency conditions).
The necessity for determining the terminal portion of the opposite strands of a nucleic acid sequence of interest and preparing two primers hybridizable thereto may be avoided by means of a kit marketed by Clontech Laboratories of Palo Alto, California (7). The Clontech UNI-AMPTM Adaptor (Cat. No. 5991-1, 5992-1, 5993-1, 5994-1 or 5995-1) is ligated onto blunt-ended DNA or cDNA of interest. A single, complementary UNI-AMP.TM. primer (Cat. No. 5990-1) is then used to amplify the DNA by the standard PCR process. Thus, by means of an attachable pre-prepared oligonucleotide adaptor, and a pre- prepared primer complementary to the adaptor, the equivalent of a conventional PCR may be performed using only one primer sequence and without any need to analyze the terminal sequences and prepare two primers. However, this method provides no specificity for the amplification. All DNA sequences present will receive a universal primer binding site and be amplified or detected by the universal primer. Thus, the universal nature of this method lacks the specificity inherent in methods which provide for specific primers designed to be complementary to a portion of the DNA sequence of interest.
Most recently, Caetan-Anolles et al. have reported DNA amplification fingerprinting using very short arbitrary primers (7). By reducing the primer size to a range of five to nine bases, Caetan-Anolles et al. were able to relax the stringency of the polymerase reaction. A characteristic spectrum of short DNA products of varying complexity was produced with 30-40 thermal cycles. The reported mechanism was priming at multiple, unspecified priming sites on each DNA target sequence. The major disadvantages of this reported single primer system are its complete lack of specificity and the consequent mixture of short amplification products. The Caetan-Anolles et al. assay does not possess the advantages of a method for single primer amplification which is specific for target nucleic acid sequences of interest. The requirement for two primers, each complementary to an opposite terminal of a polynucleotide sequence of interest, to achieve the exponential amplification of PCR represents a relative disadvantage to the artisan seeking a lower cost, simple and rapid method of practicing in vitro amplification of nucleic acid at an exponential rate. The disadvantages of two-primer PCR include the necessity of preparing two oligonucleotide primers, and, as described above, the necessity of confirming that the paired primers do not participate in unplanned hybridizations, including avoiding complementarity between the two primers (especially at the 3' end) to prevent them from linking and forming a template able to overwhelm the reaction by replicating primer dimers (resulting in an artifact which can seriously interfere with PCR results) (8). There is another potential source of the primer dimer artifact. The tag DNA polymerase and certain other polymerases have been shown to have a weak non-template directed activity which can attach bases to a blunt-ended duplex (8,9). It has been hypothesized that if this non- template directed activity were to occur on a single-stranded oligonucleotide, there is a good chance that the extension would form a short 3' overlap with the other primer which could promote dimerization (8). When primer dimers have been analyzed, they have been found to be composed of both primers. Thus, one way to avoid primer dimerization would be to use only a single unpaired primer.
Two-primer amplification has been used to isolate new gene sequences from a polynucleotide sequence library. However, the requirement for primers complementary to the sequences of the opposite termini of both strands of the new gene sequence has represented a real obstacle to the use of polymerase amplification for this purpose. of course it is well known that a new gene may be isolated by means of a sufficiently complementary probe incorporating a portion of the sequence of the new gene, but probe isolation methods lack the sensitivity provided by PCR.
A similar limitation exists in the practice of PCR to provide amplification of multiple nucleic acid sequences of interest present in the same sample. In addition, as the number of primers present in an amplification mixture increases, the efforts required to avoid unplanned hybridization between each primer and the nucleic acid sequence of interest (target sequences), or between two or more primers (e.g. resulting in primer dimer artifacts), greatly increases.
Thus, it can be appreciated that a heretofore unavailable method for achieving exponential amplification of a specific nucleic acid sequence of interest requiring only a single primer but retaining specificity of action would be an important and unexpected contribution to the art.